This work has been supported&#160;by ANR (Agence National de la Recherche) Grant ANR- 17-CE14-0001-1.

Control human samples were obtained from volonteer blood donors who gave written informed consent recruited by the blood transfusion center where the research was performed (Etablissement Fran&#231;ais du Sang-Grand Est). All procedures were registered and approved by the French Ministry of Higher Education and Research and registered under the number AC_2015_2371.The donors gave their approval in the CODHECO number AC- 2008 - 562 consent form, in order for the samples to be used for research purposes. Human studies were...

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+megakaryocytopoiesis+and+thrombopoiesis+by+the+c-Mpl+ligand.">Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand.</a> Nature. 369 (6481), 533-538 (1994).</li><li>Almomani, M. H., Mangla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> StatPearls. , (2020).</li><li>Strassel, C., Hechler, B., Bull, A., Gachet, C., Lanza, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+mice+lacking+the+GPIb-V-IX+complex+question+the+role+of+this+receptor+in+atherosclerosis.">Studies of mice lacking the GPIb-V-IX complex question the role of this receptor in atherosclerosis.</a> Journal of Thrombosis and Haemostasis. 7 (11), 1935-1938 (2009).</li><li>Delalat, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+ex+vivo+expansion+of+human+umbilical+cord+blood-derived+CD34++stem+cells+and+their+cotransplantation+with+or+without+mesenchymal+stem+cells.">Isolation and ex vivo expansion of human umbilical cord blood-derived CD34+ stem cells and their cotransplantation with or without mesenchymal stem cells.</a> Hematology. 14 (3), 125-132 (2009).</li><li>Yin, T., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stem+cell+niches+in+bone.">The stem cell niches in bone.</a> The Journal of Clinical Investigation. 116 (5), 1195-1201 (2006).</li><li>Salunkhe, V., Papadopoulos, P., Guti&#233;rrez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+megakaryocytes+from+human+peripheral+blood+mononuclear+cells.">Culture of megakaryocytes from human peripheral blood mononuclear cells.</a> Bio-protocol. 5 (21), 1639 (2015).</li><li>Peytour, Y., Villacreces, A., Chevaleyre, J., Ivanovic, Z., Praloran, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discarded+leukoreduction+filters:+a+new+source+of+stem+cells+for+research,+cell+engineering+and+therapy.">Discarded leukoreduction filters: a new source of stem cells for research, cell engineering and therapy.</a> Stem Cell Research. 11 (2), 736-742 (2013).</li><li>Lapostolle, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repopulating+hematopoietic+stem+cells+from+steady-state+blood+before+and+after+ex+vivo+culture+are+enriched+in+the+CD34(+)CD133(+)CXCR4(low)+fraction.">Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in the CD34(+)CD133(+)CXCR4(low) fraction.</a> Haematologica. 103 (10), 1604-1615 (2018).</li><li>Ivanovic, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-blood+leuko-depletion+filters+as+a+source+of+CD+34++progenitors+potentially+usable+in+cell+therapy.">Whole-blood leuko-depletion filters as a source of CD 34+ progenitors potentially usable in cell therapy.</a> Transfusion. 46 (1), 118-125 (2006).</li><li>Strassel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aryl+hydrocarbon+receptor-dependent+enrichment+of+a+megakaryocytic+precursor+with+a+high+potential+to+produce+proplatelets.">Aryl hydrocarbon receptor-dependent enrichment of a megakaryocytic precursor with a high potential to produce proplatelets.</a> Blood. 127 (18), 2231-2240 (2016).</li><li>Do Sacramento, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+human+platelets+derived+in+vitro+from+CD34(+)+cells.">Functional properties of human platelets derived in vitro from CD34(+) cells.</a> Scientific Reports. 10 (1), 914 (2020).</li><li>Blin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+model+of+the+platelet-generating+organ:+beyond+bone+marrow+biomimetics.">Microfluidic model of the platelet-generating organ: beyond bone marrow biomimetics.</a> Scientific Reports. 6, 21700 (2016).</li><li>Ito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turbulence+activates+platelet+biogenesis+to+enable+clinical+scale+ex+vivo+production.">Turbulence activates platelet biogenesis to enable clinical scale ex vivo production.</a> Cell. 174 (3), 636-648 (2018).</li><li>Pallotta, I., Lovett, M., Kaplan, D. L., Balduini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+system+for+the+in+vitro+study+of+megakaryocytes+and+functional+platelet+production+using+silk-based+vascular+tubes.">Three-dimensional system for the in vitro study of megakaryocytes and functional platelet production using silk-based vascular tubes.</a> Tissue Engineering. Part C, Methods. 17 (12), 1223-1232 (2011).</li></ol>

This protocol describes a method for producing MK capable of emitting proplatelets from blood-derived HP and to release platelets from the culture medium. HP are obtained from LRF, a by-product of the blood banks, used to remove contaminating leukocytes from cellular blood products and avoid adverse reactions. Although this method is relatively simple, a few points deserve special attention.
Deposition of the cell suspension on the density gradient medium (step 1.3.1) has to be performed gentl...

Blood platelets come from specialized large polyploid cells, the megakaryocytes (MK), that originate from a constant and fine-tuned production process known as megakaryopoiesis (MKP). At the apex of this process are hematopoietic stem cells which, in contact with the bone marrow environment (cytokines, transcription factors, hematopoietic niche), will be able to proliferate and differentiate into hematopoietic progenitors (HP) able to commit toward the megakaryocytic pathway, giving rise to immature MKs1. Under the influence of various cytokines, and in particular thrombopoietin (TPO), which is the major cytokine of MKP; the MK will then undergo two major stages of maturation: endomitosis and the development of demarcation membranes (DMS). This fully mature MK then appears close to a sinusoid vessel in which it can emit cytoplasmic extensions, the proplatelets, which will be released under the blood flow and subsequently remodeled into functional platelets2. The cloning of TPO in 19943 provided a boost in the study of MKP by accelerating the development of in vitro culture techniques allowing HP differentiation and MK maturation.
There are many pathologies affecting blood platelets, both in terms of platelet number (increase or decrease) and function4,5. Being able to recapitulate MKP in vitro from human HP could improve understanding of the molecular and cellular mechanisms underlying this process and ultimately the therapeutic management of patients.
Various sources of human HP are suitable: cord blood, bone marrow, and peripheral blood6,7,8. Harvesting HP from peripheral blood raises less logistical and ethical problems than their recovery from cord blood or the bone marrow. HP can be recovered from leukapheresis or buffy coat, but these sources are expensive and not always available in blood centers. Other protocols, less expensive and easier to perform, allow direct recovery of human peripheral blood mononuclear cells (PBMCs) without the need for prior CD34 driven isolation4,8. However, the purity of megakaryocytes is not satisfactory with this method and a selection of CD34+ cells from PBMC is recommended for optimal differentiation into MK. This led us to implement a HP purification from leukoreduction filters (LRF), routinely used in blood banks to remove white blood cells and thus avoid adverse immunological reactions9. Indeed, since 1998, platelet concentrates have been automatically leukodepleted in France. At the end of this process, LRF are discarded and all the cells retained in the LRF are destroyed. Cells in LRFs are, therefore, readily available at no additional cost. LRFs have a cellular content close to that obtained by leukapheresis or in buffy coats, notably in their composition of CD34+ HP making them a remarkably attractive source10. LRF as a human HP source has already been demonstrated to provide cells with intact functional capacities11. This source has the advantage of being abundant and affordable for laboratory research. In this context, this article describes successively: i) the extraction and selection of CD34+ HP from LRFs; ii) a two-phase optimized culture, which recapitulates the commitment of HP into the megakaryocytic pathway and the maturation of MK capable of emitting proplatelets; iii) a method for efficiently releasing platelets from these MK; and iv) a procedure for phenotyping MK and cultured platelets.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >7-AAD</td>
			<td >Biolegend</td>
			<td >558819</td>
			<td ></td>
		</tr>
		<tr >
			<td >ACD</td>
			<td>EFS-Alsace</td>
			<td >NA</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD34-PE&#160;</td>
			<td>Miltenyi biotec</td>
			<td>130-081-002</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD34-PECy7</td>
			<td >eBioscience</td>
			<td >25-0349-42</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD41-Alexa Fluor 488</td>
			<td >Biolegend</td>
			<td >303724</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD42a-PE</td>
			<td >BD Bioscience</td>
			<td >559919</td>
			<td></td>
		</tr>
		<tr >
			<td >Apyrase</td>
			<td >EFS-Alsace</td>
			<td >NA</td>
			<td></td>
		</tr>
		<tr >
			<td >BD Trucount Tubes</td>
			<td >BD Bioscience</td>
			<td>340334</td>
			<td></td>
		</tr>
		<tr >
			<td >CD34 MicroBead Kit UltraPure, human&#160;</td>
			<td>Miltenyi biotec</td>
			<td>130-100-453</td>
			<td></td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td>Heraeus</td>
			<td>Megafuge 1.OR</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Compteur ADAM&#160;</td>
			<td>DiagitalBio</td>
			<td >NA</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Cryotubes</td>
			<td>Dutscher</td>
			<td>55002</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Dextran from leuconostoc spp&#160;</td>
			<td>Sigma</td>
			<td>31392-50g</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >DMSO Hybri-max&#160;</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr >
			<td >EDTA 0.5 M&#160;</td>
			<td>Gibco</td>
			<td>15575-039</td>
			<td></td>
		</tr>
		<tr >
			<td >Eppendorf 1,5 mL&#160;</td>
			<td>Dutscher</td>
			<td>616201</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Filtration unit Steriflip PVDF</td>
			<td>Merck Millipore Ltd</td>
			<td>SE1M179M6</td>
			<td></td>
		</tr>
		<tr >
			<td >Flow Cytometer</td>
			<td >BD Bioscience</td>
			<td >Fortessa</td>
			<td></td>
		</tr>
		<tr >
			<td >Human LDL</td>
			<td>Stemcell technologies</td>
			<td>#02698</td>
			<td></td>
		</tr>
		<tr >
			<td >ILOMEDINE 0,1 mg/1 mL</td>
			<td >Bayer</td>
			<td>MA038EX</td>
			<td></td>
		</tr>
		<tr >
			<td >Inserts</td>
			<td>Fenwal</td>
			<td>R4R1401</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Laminar flow hood&#160;</td>
			<td>Holten</td>
			<td >NA</td>
			<td>Archived product</td>
		</tr>
		<tr >
			<td >LS Columms&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Lymphoprep</td>
			<td>Stemcell</td>
			<td>7861</td>
			<td></td>
		</tr>
		<tr >
			<td >Pen Strep Glutamine (100x)</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS (-)</td>
			<td>Life Technologies</td>
			<td>14190-169&#160;</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >PGi2</td>
			<td >Sigma</td>
			<td >P6188</td>
			<td></td>
		</tr>
		<tr >
			<td >Poches de transferts 600ml&#160;</td>
			<td>Macopharma</td>
			<td>VSE4001XA</td>
			<td></td>
		</tr>
		<tr >
			<td >Pre-Separation Filters (30&#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr >
			<td >StemRegenin 1 (SR1)</td>
			<td>Stemcell technologies</td>
			<td>#72344</td>
			<td></td>
		</tr>
		<tr >
			<td >StemSpan Expansion Supplement (100x)</td>
			<td>Stemcell technologies</td>
			<td>#02696</td>
			<td></td>
		</tr>
		<tr >
			<td >StemSpan-SFEM&#160;</td>
			<td>Stemcell technologies</td>
			<td>#09650</td>
			<td></td>
		</tr>
		<tr >
			<td >Stericup Durapore 0,22&#181;m PVDF</td>
			<td>Merck Millipore Ltd</td>
			<td>SCGVU05RE</td>
			<td></td>
		</tr>
		<tr >
			<td >SVF Hyclone&#160;</td>
			<td>Thermos scientific</td>
			<td>SH3007103</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringues 30 mL&#160;</td>
			<td>Terumo</td>
			<td>SS*30ESE1</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Syringe filters Millex 0,22&#181;M PVDF</td>
			<td>Merck Millipore Ltd</td>
			<td>SLGV033RB</td>
			<td></td>
		</tr>
		<tr >
			<td >TPO</td>
			<td>Stemcell technologies</td>
			<td>#02822</td>
			<td></td>
		</tr>
		<tr >
			<td >Tubes 50 mL</td>
			<td>Sarstedt</td>
			<td>62.548.004 PP</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Tubes 15 mL&#160;</td>
			<td>Sarstedt</td>
			<td>62.554.001 PP</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Tubulures</td>
			<td>B Braun</td>
			<td>4055137</td>
			<td>Or equivalent material</td>
		</tr>
	</tbody>
</table>

leukodepletion filters-derived cd34+ cells as a cell source to study megakaryocyte differentiation and platelet formation

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing platelets allows an in-depth study of the mechanisms underlying platelet biogenesis. Available culture protocols are mostly based on hematopoietic progenitors derived from bone marrow or cord blood raising a number of ethical, technical, and economic concerns. If there are already available protocols for obtaining CD34 cells from peripheral blood, this manuscript proposes a straightforward and optimized protocol for obtaining CD34+ cells from leukodepletion filters readily available in blood centers. These cells are isolated from leukodepletion filters used in the preparation of blood transfusion products, corresponding to eight blood donations. These filters are meant to be discarded. A detailed procedure to collect hematopoietic progenitors identified as CD34+ cells from these filters is described. The method to obtain mature megakaryocytes extending proplatelets while discussing their phenotypic evolution is also detailed. Finally, the protocol present a calibrated pipetting method, to efficiently release platelets that are morphologically and functionally similar to native ones. This protocol can serve as a basis for evaluating pharmacological compounds acting at various steps of the process to dissect the underlying mechanisms and approach the in vivo platelet yields.

Extraction and selection of CD34+ cells from LRFs 
Here, the method, derived from Peytour et al.9, describes the extraction and selection of CD34+ cells from discarded LRFs available in blood banks after leukocyte removal. Following the backflush procedure, usually 1.03 x 109 &#177;&#160;2.45 x 108 cells/LRF (Mean&#177;SEM; n = 155) are recovered with a viability of 94.88 &#177; 0.10% (Figure 2A i...

Watch this Scientific Journal Video about Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation at JoVE.com

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation

This protocol describes in detail all the steps involved in obtaining leukofilter-derived CD34+ hematopoietic progenitors and their in vitro differentiation and maturation into proplatelet-bearing megakaryocytes that are able to release platelets in the culture medium. This procedure is useful for in-depth analysis of cellular and molecular mechanisms controlling megakaryopoiesis.

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing ...

This protocol proposes a simple method for obtaining human hematopoietic progenitors, and for inducing their differentiation into megakaryocytes. It can serve as a basis for better understanding of megakaryopoiesis This technique uses a source, but offers the advantage of being affordable and abundant for laboratory research in hematopoiesis. This method is used to better understand megakaryopoiesis for the cell to be used in therapy they will need to be prepared in the GMP conditions, which is currently not the case.Understanding the steps including PBMC collection can only be fully complete with a visual demonstration. Before beginning an experiment, connect a leukoreduction filter to an empty 600 milliliter transfer bag and to a tubing set. In a bio safety cabinet inject 25 milliliters of filtered elution buffer per leukoreduction filter into the empty bag, and use a 30 milliliter syringe to gently aspirate the bag contents through the filter.Transfer the collected red blood cells into a new 50 milliliter tube, and dilute the cell suspension by half with 2%dextran. Mix well to aggregate the blood cells and allow the cells to sediment for 30 minutes at room temperature. When the red blood cells have settled transfer the supernatant to a 50 milliliter tube and fill the tube with two millimolar PBS EDTA solution.Gently overlay half of the supernatant onto 25 milliliters of density gradient medium in each of two 50 milliliter tubes without disturbing the gradient surface and separate the cells by density gradient centrifugation. Use a disposable pipette to transfer the cells from each interface to a new 50 milliliter tube. Wash the cells two times in 50 milliliters of two millimolar PBS EDTA per wash.After the second wash, resuspend the pellets in a single 50 milliliter volume of fresh PBS EDTA for counting. Here is possible to stop the procedure by maintaining the collected cells under agitation at four degrees Celsius during the night. For CD34+cell selection, determine the cell number and collect the cells by centrifugation and resuspend the cell pellet in 300 microliters of two millimolar PBS EDTA per 10 to the eighth cells.Add the appropriate volume of FC receptor blocking reagent and 50 microliters of CD34 micro-beads per 10 to the eighth cells. After 30 minutes at four degrees Celsius, wash the cells with two millimolar PBS EDTA, and resuspend the pellet in 500 microliters of fresh two millimolar PBS EDTA, per 10 to the eight cells. Humidify and appropriately size magnetic column with PBS EDTA, and then add the sample to the column.Wash the column two times with three milliliters of two millimolar PBS EDTA per wash. Before eluding the CD34+cells, with two five milliliter volumes of two millimolar PBS EDTA per elution. To assess the purity of the magnetic bead selected cells, add two microliters of an appropriate human anti CD34 antibody to a 100 microliter aliquot of the isolated cells and mix well before incubating for 15 minutes at four degrees celsius.Following incubation, wash the cells in PBS and resuspend the pellet in 200 microliters of PBS for analysis of the purity of the cell sample by flow cytometry. After counting, collect the CD34+cells by centrifugation and immediately resuspend the pellet in cold solution one to a density of 10 to the six cells per milliliter before quickly adding the cell suspension to cold solution two. Then immediately place the cryo tube in a minus 80 degree Celsius freezer for 24 hours before transferring the cryo tube to liquid nitrogen storage.In this representative study, the cells were recovered by leukoreduction filtration as demonstrated with an approximate 95%viability. After magnetic bead selection, a greater than 90%pure CD34+cell population was obtained. Cell proliferation typically decreases after a week of culture, but with no significant changes in cell viability.By day seven, the CD34+cells should begin expressing CD41, a specific and early marker or for megakaryocyte and platelet development. By day 10, the majority of the cells in the culture typically develop into mature CD41 expressing megakaryocytes. By day 13, megakaryocyte pro platelet extension and platelet release can be observed by light microscopy.The total number of CD41, CD42, 8 positive platelets released into the culture can then be quantified using a calibrated number of fluorescent beads. This method paves the way for enhancing the underlying mechanisms of megakaryopoiesis and for increasing platelets yields in visual. Even if this method seems tedious, it is very simple you just need to be patient and prepare all the regions in advance.Although our critical concerns megakaryopoiesis we obtain CD42 cells can be used in over hematopoietic pathways.

leukodepletion-filters-derived-cd34-cells-as-cell-source-to-study

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Biology

3.7K vistas.  Université de Strasbourg, INSERM, EFS Grand Est. Este protocolo propone un método sencillo para la obtención de progenitores hematopoyéticos humanos, y para inducir su diferenciación en megacariocitos. Puede servir como base para una mejor comprensión de la megacaropoyesis Esta técnica utiliza una fuente, pero ofrece la ventaja de ser asequible y abundante para la investigación de laboratorio en hematopoyesis. Este método se utiliza para comprender mejor la megacaropoyesis para que la célula se utilice en la terapia, deberá prepar...